This invention relates to the generation of daughter ion spectra from analyte substances that are ionized by matrix assisted laser desorption. For the purposes of the ionization of analyte ions by matrix assisted laser desorption, the samples, consisting mainly of matrix substance with a small number of embedded analyte molecules, are exposed to short pulses of light from a UV laser. Each pulse of laser light generates a plasma cloud. When the pulses of laser light have only moderate power, from the analyte substances practically only molecular ions are created, no fragment ions; therefore several types of analyte substance can be present and recognized in the sample simultaneously—in other words, mixture analyses can be carried out. Predominantly, however, complex ions of decomposed and modified matrix substances are also generated. The creation of the analyte and matrix ions in the laser-generated plasma is very intricate, and not every aspect is yet understood. Although the matrix substances have molecular weights in the range of between only 150 and 300 Daltons, the plasma contains many complex ions composed primarily of fragments of matrix molecules of such varied masses that, in the range up to about 1000 Daltons, almost every mass number in the mass spectrum is occupied by multiple ions of different compositions.
The method of ionization by matrix assisted laser desorption is primarily used to investigate large biomolecules, particularly large biopolymers such as proteins or peptides obtained from proteins by enzymatic digestion, which yield mass spectra that can be evaluated effectively above 1000 Daltons, so that the background noise does not prevent the evaluation. It is also possible to investigate conjugates of peptides with sugars (glycopeptides) or fats (lipopeptides).
By recording the mass spectra of daughter ions obtained through deliberate fragmentations of the analyte ions, the protein sequences, and also the structures of the conjugates, can be analyzed. Two different kinds of fragmentation can be carried out in special MALDI time-of-flight mass spectrometers in order to generate daughter ions and, particularly in the case of proteins and peptides, they lead to different fragmentation patterns. The two types of fragmentation are referred to as ISD (“in-source decay”) and PSD (“post-source decomposition”).
To record daughter ion spectra created by PSD, the intensity of the laser light is increased. As a result, a large number of unstable analyte ions are created which, after their acceleration in the mass spectrometer, decompose with characteristic half-lives, so forming daughter ions (also known as fragment ions). The unstable ions which decompose in the flight path of the mass spectrometer are referred to as “metastable” ions. Increasing the intensity of the laser light, however, increases not only the number of metastable analyte ions but also the number and size of the matrix-containing complex ions, which now cover masses of up to 3000 Daltons and above. Recording the PSD daughter ion spectra is at present done in time-of-flight mass spectrometers specially designed for this purpose, such as are described in detail in patent DE 198 56 014 C2 (C. Köster et al., corresponding to GB 2 344 454 B and U.S. Pat. No. 6,300,627 B1).
FIG. 1 schematically illustrates a MALDI state-of-the-art time-of-flight mass spectrometer of this type for recording daughter ion spectra. A UV pulsed laser (3) sends a pulse of laser light through a focusing lens (4) and a deflecting mirror (5) onto the sample (6), which is located on a sample support (1) in a solid state obtained by drying a droplet of sample solution. A small amount of the sample material abruptly evaporates, creating a plasma cloud. Accelerating potentials at the acceleration diaphragms (7) and (8) form the ions in the plasma cloud into an ion beam (9); moderate accelerating voltages give the ions that will be used for recording the daughter ion spectra a relatively low energy of only, for instance, 6 keV. Switching on the accelerating voltage with a delay relative to the flash of laser light provides time-focusing of the ions at the location of the parent ion selector (10), improving selection. This parent ion selector is a bipolar switchable grid which only allows ions through during an adjustable switching time window, so making them available for further analytical investigation. The parent ion selector is thus used to select the parent ions whose daughter ions are to be measured. If metastable parent ions have already decomposed between the acceleration diaphragm (8) and the parent ion selector (10), the daughter ions created here can also pass through the parent ion selector, as they have the same velocity as the undecomposed parent ions, and therefore arrive at the parent ion selector at the same time as the latter arrive.
The undecomposed parent ions and the daughter ions that have been created through the decomposition of parent ions, now fly on to a post-acceleration unit (12), where they are given an additional acceleration by about 20 kilovolts. Prior to the post-acceleration, the daughter ions only possess a fraction of the energy of the parent ions, corresponding to their mass fraction relative to the parent ion. The post-acceleration now gives the daughter ions an energy of between 20 and 26 keV, which is particularly favorable for an analysis of their energy—and therefore of their mass—in the reflector (14). The energy analysis, in turn, is carried out by analyzing the time-of-flight at the detector (17), since the lighter ions, even if lower in energy, are faster and also reach the detector more quickly along the shorter beam (15) than the more energetic, but slower, ions traveling along the beam (16) that enters more deeply into the reflector (14).
In order that those daughter ions created by decomposition of the post-accelerated parent ions that have not yet decomposed cannot reach the reflector (14), a further ion selector (13) is included in the ion path between the post-acceleration unit (12) and the reflector (14) to suppress the parent ions and their equally fast daughter ions. This parent ion suppressor is not only necessary to suppress the daughter ions created after the post-acceleration, but also to suppress the continuous background that would be generated by the daughter ions from parent ions that decompose further at an undetermined potential in the reflector.
In this modern PSD method for recording daughter ion spectra, it is therefore necessary to select the parent ions whose daughter ion spectra are to be recorded. However, not only the parent ions are selected by means of the switchable grid in the parent ion selector (10) during the switched time window, but also a large number of the extraordinarily frequent matrix-containing complex ions, or the fragment ions that have formed from them, provided only that the complex ions have the correct mass and therefore arrive at the parent ion selector within the correct time window. These fragment ions, formed from the complex ions, result in a background noise signal which, by raising the noise, lowers the sensitivity.
If the complex ions contain relatively large, stable molecule fragments, such as analyte ions from the analyte mixture that are not to be selected at all, ghost signals can occur. It has been observed, for example, that the molecular ions of other types of analyte ion from the sample that were not selected as parent ions appeared in the daughter ion spectra. These molecular ions could only have attained a mass equal to that of the selected parent ions by complexing with matrix fragments. In this way they can pass through the parent ion selector, and are then measured in the daughter ion spectrum, if decomposed back into analyte ions and the associated complex of matrix fragments. It must here be emphasized yet again that these ghost signals can also be measured if the complex ions decompose soon after full acceleration, but at a point that is still distant from the parent ion selector.
It appears possible that a high proportion of the analyte ions are created in a way that temporarily includes such a complex state. It is entirely possible that a matrix complex ion attaches to a neutral analyte molecule, transfers a proton to the analyte molecule, and splits off again after a rearrangement and stabilization time. It is also possible to transfer additional energy to the analyte molecule, with the result that it then becomes metastable and can decompose further at a later stage. The lifetime of these complexes is not known. If such a complex ion consisting of an unwanted analyte ion with attached matrix molecule fragments happens to have exactly the mass of the parent ions that are to be selected, and if it survives the acceleration in the ion source, it will be included in the selection made by the parent ion selector, and can lead to ghost signals when it decomposes. It is most probable that the associated decomposition will occur a long way upstream of the parent ion selector.
If, on the other hand, the complex ions already decompose in the acceleration region, this will yield ions of lower, undefined velocity. These ions constitute a high proportion of the undefined, smeared background of every MALDI mass spectrum. A proportion of these ions reaches the parent ion selector at exactly the time when it is open in order to select the parent ions. Whether or not these ions then decompose further, they create a more or less continuous background in the daughter ion spectra, smeared across all the masses in the mass spectrum.
If the complex ions that contain an analyte molecule decompose prior to the acceleration, that is to say in the delay phase before the acceleration is switched on, into an analyte molecule and the attached remainder, these analyte ions can contribute to the analysis quite normally. Their mass and charge is identical to the ions originally created in the plasma. Once again, a large number of metastable analyte ions can result.
Metastable ions of the same type but different genesis do not have a consistent half-life. Rather, their half-life depends on the internal energy that they have absorbed in the plasma or in complexing processes. It is not known whether the type of decomposition, that is the fragmentation pattern of the bonds between the individual molecule parts, also depends on the quantity of internal energy. All that is known is that the spontaneous fragmentation of protein ions in a time range of less than 10−8 seconds (ISD) demonstrates a remarkably different fragmentation pattern from the fragmentation of the metastable ions (PSD) decaying in a time range greater than 10−5 seconds. The spontaneous fragmentation (ISD) can be classified as an “electron-induced” type of fragmentation, whereas the slow fragmentation (PSD) is regarded as “ergodic” fragmentation, which, in principle, requires a balanced internal distribution of the energy across the individual vibration states. It is not known whether there is an intermediate state with mixed fragmentation patterns.
The degree to which the decomposition half-life of metastable ions depends on their mass and the internal structure is also unknown. There are, however, some indications that metastable complex ions have very short half-lives and decompose very quickly, the great majority doing so before reaching the parent ion selector.
As was already explained above, there is a second type of fragmentation (ISD) that can be exploited for recording daughter ion spectra. It does not, however, play any role in this invention. It is based on the fact that the ions also fragment spontaneously in the laser plasma. If a sample that contains only one analyte substance at a suitable concentration is exposed to a pulse of laser light of high intensity, fragment ions of the analyte substance form within a period of less than 10−8 seconds. Due to the delay prior to the start of acceleration, these fragment ions are only accelerated after they have been formed, and can therefore be measured in a mass spectrum recorded in the normal way. This type of daughter ion formation is called ISD (“in-source decomposition”).
The term “mass” here always refers to the “mass-to-charge ratio” m/z, which alone is relevant for mass spectrometry, and not simply the “physical mass”, m. The dimensionless number z represents the number of elementary charges on the ion, that is the number of excess electrons or protons on the ion that have an external effect as an ion charge. Without exception, all mass spectrometers can only measure the mass-to-charge ratio m/z, not the physical mass m itself. The mass-to-charge ratio is the mass fraction per elementary charge on the ion. Correspondingly, “light” or “heavy” ions always refers to ions with a low or high mass-to-charge ratio m/z. The term “mass spectrum” again always refers to the mass-to-charge ratios m/z.